A Gd MOF composite material, an immunosensor, its preparation method and application

Abstract

The application relates to the technical field of immunosensors, in particular to a Gd MOF composite material, an immunosensor and a preparation method and application thereof. The preparation method of the Gd MOF composite material comprises the following steps: dissolving polyether F127 in a mixed solvent, then adding gadolinium (Gd) salt and 5-aminoisophthalic acid (5-AIPA) to carry out a first heating reaction to obtain Gd MOF; dispersing the Gd MOF in a copper salt solution to carry out a second heating reaction to obtain the Gd MOF composite material. The application further provides a double-ligand Gd MOF composite material prepared by the method, a luminescent body combined with an antibody, an immunosensor and a preparation method. The application provides a novel MOF material, and the MOF material is used in an ECL sensing system, so that efficient detection of carbohydrate antigen 19-9 (CA19-9) is realized.

Description

Technical Field

[0001] This invention relates to the field of immune sensor technology, specifically to a Gd MOF composite material, an immune sensor, its preparation method, and its application. Background Technology

[0002] Electrogenerated chemiluminescence (ECL) refers to the process by which active materials on the electrode surface or in solution undergo a series of redox reactions under the influence of an applied voltage, generating an excited-state luminescent material. When this excited state returns to the ground state, energy is released in the form of photons, thus achieving optical signal output. Because it does not require an external excitation source, ECL has advantages such as low background, high sensitivity, good controllability, and high spatiotemporal resolution, and has been widely used in fields such as bioanalysis, environmental monitoring, food safety, and clinical diagnostics.

[0003] As a key component of the ECL system, the performance of the luminescent material directly determines the sensitivity, stability, and selectivity of the sensing platform, and therefore has been extensively studied by researchers. Classical luminescent materials include ruthenium bipyridine and its derivatives, luminol, quantum dots, semiconductors, and metal nanoclusters. It is well known that lanthanide materials possess excellent optical properties such as long luminescence lifetime, large Stokes shift, and high quantum yield, and some lanthanide metals have been extensively studied as "star elements."

[0004] Metal-organic frameworks (MOFs) are a new class of highly crystalline and porous materials composed of metal ions or metal clusters linked to organic ligands. They possess characteristics such as large surface area, high framework flexibility, tunable porosity, and customizability, and have been widely applied in gas separation and adsorption, luminescence, chemical catalysis, drug delivery, chemical sensing, energy storage, and conversion. Lanthanide MOFs combine the advantages of both, with the luminescent properties of lanthanide ions and the tunable topology of MOFs laying the foundation for the development of luminescent materials with specific applications.

[0005] Based on this, lanthanide MOFs can be used to construct unique ECL sensing platforms. Many related sensors have been developed and reported in the literature for detecting cations, anions, small molecules, biomolecules, and explosive chemicals. For example, 3D lanthanide MOFs (Tb MOFs) are used as the core material for luminescent sensors to detect paraquat, and this material exhibits strong water stability and resistance to acids and alkalis. Even in harsh environments with common pollutants, it can rapidly detect paraquat in the environment and agricultural products with high sensitivity and selectivity. Another example is the dual-emission lanthanide metal-organic framework (Eu... 0.07 Gd 0.03For example, the ECL behavior of metal-organic frameworks with Eu as the central atom / ion has opened up new possibilities for the application of lanthanide MOF materials in the ECL field. Furthermore, a novel luminescent metal-organic framework, named Ce-TCPP-LMOF, was designed using the emerging ECL material (4,4',4",4‴-(porphyrin 5,10,15,20-tetramethyl)tetrabenzoic acid) as an organic ligand and Ce(III) as a metal node, showing promising application prospects. In addition, a signal-amplifying ECL sensor chip has been developed using Ce(III, IV) MOF as a co-reaction promoter in the isoluminol-dissolved oxygen system for the sensitive analysis of procalcitonin.

[0006] Although there have been ECL studies on Eu, Tb, and Ce-based Ln-MOFs, gadolinium (Gd), as a lanthanide ion, has not yet been publicly reported in the ECL field for its MOF materials. Therefore, developing ECL sensing systems with Gd-MOFs as the core luminescent material or functional promoter can not only fill the technological gap in this area but also break through the current bottlenecks in sensitivity and selectivity, providing a new generation of high-performance ECL platforms for environmental monitoring, bioanalysis, and medical diagnostics. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a Gd MOF composite material, an immune sensor, a method for preparing the same, and its application, so as to provide a novel MOF material for use in ECL sensing systems, thereby filling a technological gap in this field.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a Gd MOF composite material includes the following steps:

[0010] Polyether F127 was dissolved in a mixed solvent, and then gadolinium (Gd) salt and 5-aminoisophthalic acid (5-AIPA) were added to carry out the first heating reaction to obtain Gd MOF;

[0011] Gd MOF was dispersed in a copper salt solution and subjected to a second heating reaction to obtain a Gd MOF composite material, namely Cu:GdMOF. Here, Cu:Gd MOF represents a Cu-doped Gd MOF composite material.

[0012] Based on the above technical means, Cu:Gd MOF was obtained by using Gd(III) as the central metal ion, 5-aminoisophthalic acid (5-AIPA) as the organic ligand, and doping Cu into Gd MOF. It was determined that when the Gd MOF composite material is used in the ECL sensing system, it can act as a self-reinforced light emitter, effectively shorten the charge transfer distance and accelerate the electron transfer rate, and greatly enhance the ECL signal, thus filling the gap of gadolinium materials in the field of ECL sensors.

[0013] Preferably, the temperature of the first heating reaction is 120~130 °C and the time is 12~13 h.

[0014] Preferably, the temperature of the second heating reaction is 80~90 °C and the time is 4 h.

[0015] Preferably, the ratio of polyether F127, gadolinium (Gd) salt and 5-aminoisophthalic acid (5-AIPA) is 100 mg: 0.18 mmol: 0.18 mmol.

[0016] Preferably, the ratio of Gd MOF to copper salt is 20 mg: 0.2 mmol.

[0017] Preferably, the gadolinium (Gd) salt is selected from one or both of gadolinium chloride and gadolinium chloride hexahydrate (GdCl3·6H2O).

[0018] Preferably, the copper salt is selected from one or both of copper chloride and copper chloride dihydrate (CuCl2·2H2O).

[0019] Preferably, the mixed solvent is selected from a mixture of N,N-dimethylformamide (DMF) and water.

[0020] Preferably, the copper salt solution is an aqueous solution of copper salt.

[0021] The present invention also provides a Gd MOF composite material prepared by the preparation method described in the present invention.

[0022] The present invention also provides an application of the Gd MOF composite material prepared by the preparation method described in the present invention as a self-reinforced light emitter.

[0023] The present invention also provides an antibody-bound luminescent material, which is made of an antibody and a Gd MOF composite material prepared by the preparation method described in the present invention.

[0024] The present invention also provides a method for preparing the antibody-binding luminescent body described herein, comprising the following steps:

[0025] The Gd MOF composite material was added to water, followed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for a first incubation. Subsequently, it was dispersed in a PBS solution containing antibody II and incubated for another time to obtain a luminescent body that binds to the antibody.

[0026] Preferably, antibody II is selected from Ab2 antibody.

[0027] Preferably, the first incubation temperature is 4 °C and the time is 2 h.

[0028] Preferably, the first incubation is performed by shaking at a temperature of 4 °C for 2 h.

[0029] Preferably, the incubation temperature is 4 °C and the incubation time is 8 h.

[0030] Preferably, the continued incubation is performed by shaking at a temperature of 4 °C for 8 hours.

[0031] Among them, Ab2 antibody represents the antibody for detecting carbohydrate antigen 19-9.

[0032] The present invention also provides an immune sensor comprising an antibody-binding luminescent material prepared by the method described herein.

[0033] This invention also provides a method for preparing an immune sensor, comprising the following steps:

[0034] CuO@In2O3 was dissolved in a second organic solvent, and then 3-aminopropyltriethoxysilane (APTES) was added to carry out a fifth heating reaction to obtain CuO@In2O3-NH2;

[0035] CuO@In2O3-NH2 was dispersed in a PBS solution containing antibody I, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and a second incubation was performed to obtain a CuO@In2O3 solution bound to the antibody, namely CuO@In2O3-antibody I solution.

[0036] A CuO@In2O3 solution containing the bound antibody was dropped onto the electrode surface, followed by the sequential addition of bovine serum albumin (BSA) solution, CA19-9 antigen, and the luminescent solution containing the bound antibody to the electrode surface. A third incubation was then performed to obtain the immunosensor, namely the ECL immunosensor.

[0037] Preferably, antibody I is selected from Ab1 antibody.

[0038] Among them, Ab1 antibody represents the capture antibody against carbohydrate antigen 19-9.

[0039] Preferably, the second organic solvent is selected from anhydrous ethanol.

[0040] Preferably, the bovine serum albumin (BSA) solution is a PBS (pH=7.4) solution containing bovine serum albumin (BSA).

[0041] Preferably, the antibody-binding CuO@In2O3 solution is an antibody-binding CuO@In2O3 PBS (pH=7.4) solution.

[0042] Preferably, the luminescent solution containing the antibody is a PBS (pH=7.4) solution containing the luminescent antibody.

[0043] Preferably, the ratio of CuO@In2O3 to 3-aminopropyltriethoxysilane (APTES) is 500 mg: 1 mL.

[0044] Preferably, the temperature of the fifth heating reaction is 70~80 ℃ and the time is 2~3 h.

[0045] Preferably, the second incubation temperature is 4 °C and the time is 8 h.

[0046] Preferably, the second incubation is performed by shaking at a temperature of 4 °C for 8 h.

[0047] Preferably, the third incubation temperature is 4 ℃ and the time is 2 h.

[0048] Preferably, the third incubation is performed at a temperature of 4 ℃ for 2 h.

[0049] Preferably, the preparation method of CuO@In2O3 includes the following steps:

[0050] Polyvinylpyrrolidone (PVP) and copper salt are dissolved in a first organic solvent to obtain solution A;

[0051] Tristyric acid (H3BTC) was dissolved in a first organic solvent to obtain solution B;

[0052] Solution A and solution B were mixed and subjected to a third heating reaction to obtain Cu MOF;

[0053] Cu MOF was dissolved in a first organic solvent, and then In(NO3)3·xH2O and terephthalic acid (TPA) were added to carry out a fourth heating reaction to obtain Cu MOF@In MOF;

[0054] Cu MOF@In MOF was calcined to obtain CuO@In2O3;

[0055] The value of x is 4.5.

[0056] After epitaxial growth of In MOF by Cu MOF, further calcination was performed to obtain a CuO@In2O3 composite material with an internal hollow structure. The CuO@In2O3 composite material was used as the catalyst material in the ECL sensor. By utilizing the synergistic catalytic effect of variable valence metals Cu and In, as well as the high specific surface area brought by the hollow structure, the ECL signal was greatly enhanced, thereby greatly improving the detection sensitivity.

[0057] Preferably, the first organic solvent is selected from N,N-dimethylformamide (DMF).

[0058] Preferably, the temperature of the third heating reaction is 80~90 ℃ and the time is 24 h.

[0059] Preferably, the temperature of the fourth heating reaction is 120~130 ℃ and the time is 24 h.

[0060] Preferably, the calcination temperature is 500~550 ℃ and the time is 4 h.

[0061] Preferably, the preparation method of CuO@In2O3 includes the following steps:

[0062] Polyvinylpyrrolidone (PVP) and Cu(NO3)2·3H2O were dissolved in DMF to obtain solution A;

[0063] Pyromellitic acid (H3BTC) was dissolved in DMF to obtain solution B;

[0064] Solution A and solution B were mixed and stirred, then transferred to a reaction vessel for a third heating reaction. After centrifugation and separation, a precipitate was obtained. The precipitate was washed three times each with DMF and ethanol by centrifugation. The precipitate was then vacuum dried overnight to obtain a blue powder, which is Cu MOF.

[0065] Cu MOF was added to DMF, and after ultrasonic treatment, In(NO3)3·xH2O and terephthalic acid (TPA) were added. Then, the mixture was transferred to a reaction vessel for a fourth heating reaction. After centrifugation and separation, a precipitate was obtained. The precipitate was washed three times each with DMF, water and ethanol by centrifugation. The precipitate was then vacuum dried overnight to obtain Cu MOF@In MOF.

[0066] Cu MOF@In MOF was calcined in a muffle furnace to obtain CuO@In2O3.

[0067] The present invention also provides a CuO@In2O3 prepared by the above preparation method, namely a CuO composite material.

[0068] This invention also provides an application of CuO@In2O3 prepared by the method described in this invention as a catalytic material.

[0069] The present invention also provides a catalytic material that combines an antibody, which is made of an antibody and a CuO composite material prepared by the aforementioned preparation method.

[0070] This invention also provides a method for preparing an antibody-bound catalytic material, comprising the following steps:

[0071] The CuO@In2O3 was dissolved in a second organic solvent, and then 3-aminopropyltriethoxysilane (APTES) was added to carry out a fifth heating reaction to obtain CuO@In2O3-NH2;

[0072] CuO@In2O3-NH2 was dispersed in a PBS solution containing antibody I, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS), and a second incubation was performed to obtain the antibody-binding catalytic material, namely CuO@In2O3-Ab1.

[0073] Preferably, the method for preparing the antibody-conjugated catalytic material includes the following steps:

[0074] CuO@In2O3 was dissolved in anhydrous ethanol, then APTES was added, and the fifth heating reaction was carried out. After centrifugation and separation, the precipitate was obtained. The precipitate was dried and ground to obtain CuO@In2O3-NH2.

[0075] CuO@In2O3-NH2 was ultrasonically dispersed in a PBS solution containing Ab1 (antibody I), EDC, and NHS. After a second incubation, the mixture was centrifuged and separated to obtain a precipitate. The precipitate was washed with PBS to remove unbound substances, yielding the antibody-binding catalytic material, namely CuO@In2O3-Ab1. CuO@In2O3-Ab1 was then redispersed in PBS to obtain a CuO@In2O3-Ab1 solution for later use.

[0076] The present invention also provides an immune sensor comprising an antibody-binding catalytic material prepared by the method described herein.

[0077] Preferably, the method for preparing the immune sensor includes the following steps:

[0078] Polyether F127 was dissolved in a mixed solution of DMF and H2O, and then GdCl3·6H2O and 5-aminoisophthalic acid (5-AIPA) were added and sonicated until dissolved. The solution was then transferred to a stainless steel autoclave lined with polytetrafluoroethylene for the first heating reaction. After centrifugation and separation, a precipitate was obtained. The precipitate was washed three times each with DMF and ethanol by centrifugation. The precipitate was then vacuum dried overnight to obtain Gd MOF.

[0079] Gd MOF was dispersed in CuCl2·2H2O aqueous solution, and a second heating reaction was carried out. After centrifugation and separation, a precipitate was obtained. The precipitate was washed with water and then vacuum dried overnight to obtain the Gd MOF composite material, namely Cu:Gd MOF.

[0080] The Gd MOF composite material was added to water and ultrasonically dispersed evenly. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added for the first incubation. After centrifugation and separation, a precipitate was obtained. The precipitate was washed with water and then dispersed in PBS solution containing antibody II. After further incubation, centrifugation and separation, a precipitate was obtained to obtain the antibody-bound luminescent material, namely Cu:Gd MOF-Ab2.

[0081] The electrode was polished with alumina powder, and then the catalytic material solution of the antibody-binding agent was dropped onto the electrode surface. Subsequently, bovine serum albumin (BSA), CA19-9 antigen, and the luminescent solution of the antibody-binding agent were dropped onto the electrode surface in sequence, and a third incubation was performed to obtain the immune sensor.

[0082] The present invention also provides an application of the immunosensor prepared by the method described herein as an ECL immunosensor, wherein the immunosensor is used to detect carbohydrate antigen 19-9 (CA19-9).

[0083] An immunosensor was constructed using CuO@In2O3 as a substrate, Cu:Gd MOF as a signal probe, and carbohydrate antigen 19-9 (CA19-9) as a detection target. This sensor was then used as an ECL immunosensor. Performance characterization analysis during the detection process showed that the sensor has high sensitivity and good specificity, selectivity, and stability, which will effectively promote the practical clinical significance of lanthanide MOFs in ECL immunoassay.

[0084] Among them, CA19-9 is the preferred routine biomarker for the specific detection of small cell pancreatic cancer, and it has important clinical application value in the early diagnosis of pancreatic cancer.

[0085] Preferably, the detection limit of the immunosensor used as an ECL immunosensor for detecting carbohydrate antigen 19-9 (CA19-9) is 82.3 µU / mL. -1.

[0086] Preferably, the detection limit of the immunosensor used as an ECL immunosensor for detecting carbohydrate antigen 19-9 (CA19-9) is as low as 82.3 µU / mL. -1 (S / N = 3).

[0087] Preferably, the immunosensor is used as an ECL immunosensor to detect carbohydrate antigen 19-9 (CA19-9) in serum.

[0088] Preferably, the serum is a human serum sample.

[0089] The beneficial effects of this invention are:

[0090] The preparation method of the Gd MOF composite material of the present invention involves using Gd(III) as the central metal ion, 5-aminoisophthalic acid (5-AIPA) as the organic ligand, and doping Cu into Gd MOF to obtain Cu:Gd MOF. It has been determined that when the Gd MOF composite material is used in the ECL sensing system, it can act as a self-reinforcing light emitter, effectively shorten the charge transfer distance and accelerate the electron transfer rate, greatly enhancing the ECL signal, thereby filling the gap of gadolinium materials in the field of ECL sensors.

[0091] The immunosensor of the present invention, constructed using CuO@In2O3 as a substrate, Cu:Gd MOF as a signal probe, and carbohydrate antigen 19-9 (CA19-9) as a detection target, is an ECL immunosensor with high sensitivity, good specificity, selectivity and stability, and has application value in the field of immunosensor technology. Attached Figure Description

[0092] Figure 1 This is a flowchart of the preparation method for an immune sensor;

[0093] Figure 2 The image shows the characterization of the Gd MOF prepared in Example 1, where A is the full-area XPS spectrum, B is the fine XPS spectrum of the Gd 4d orbital, C and D are SEM images, and E is a TEM image.

[0094] Figure 3 The above are XPS spectra of the Cu:Gd MOF prepared in Example 1, where A is the full-area XPS spectrum, B is the fine XPS spectrum of Gd 4d orbitals before and after Cu doping, C is the fine XPS spectrum of Cu 2p orbitals, and D is the Auger electron spectrum of Cu.

[0095] Figure 4The images are infrared spectra, SEM images, and EDS spectra. Among them, A is the FT-IR spectrum of 5-AIPA, Gd MOF, and Cu:Gd MOF, B is the SEM image of Cu:Gd MOF, and C is the EDS mapping spectrum of Cu:Gd MOF.

[0096] Figure 5 The images show the UV-Vis absorption and fluorescence spectra, where A represents the UV-Vis absorption spectra of 5-AIPA, Gd MOF, and Cu:Gd MOF, B represents the fluorescence excitation spectrum of Gd MOF, and C represents the fluorescence emission spectrum of Cu:Gd MOF.

[0097] Figure 6 The images are XRD and XPS spectra. A represents the XRD spectra of In2O3 and CuO@In2O3 compared with the standard card; B represents the XRD spectra of CuO and In2O3 compared with the standard card; C represents the full-area XPS energy spectrum of CuO@In2O3; D represents the fine XPS spectrum of Cu 2p orbitals; E represents the fine XPS spectrum of In 3d orbitals; and F represents the fine XPS spectrum of O 1s orbitals.

[0098] Figure 7 The images are SEM images, TEM images, and EDS spectra. A is the SEM image of Cu MOF, B is the SEM image of Cu MOF@InMOF, C is the SEM image of CuO@In2O3, D is the TEM image of CuO@In2O3, E is the high-resolution transmission electron microscope image of CuO@In2O3, and F is the EDS mapping spectrum of CuO@In2O3.

[0099] Figure 8 The CuO-modified electrode was tested at 5.0 mmol / L. -1 CV curves at different scan rates in K3[Fe(CN)6] solution and the linear relationship between peak current and scan rate;

[0100] Figure 9 The CuO@In2O3 modified electrode was tested at 5.0 mmol / L. -1 CV curves at different scan rates in K3[Fe(CN)6] solution and the linear relationship between peak current and scan rate;

[0101] Figure 10 Cyclic voltammetry curves under electrode layer-by-layer modification;

[0102] Figure 11 The AC impedance spectrum is shown under electrode layer-by-layer modification.

[0103] Figure 12The graphs show the ECL response results under different conditions. In the graphs, A represents the ECL response results corresponding to different K2S2O8 concentrations, B represents the ECL response results corresponding to different Cu:Gd MOF concentrations, C represents the ECL response results corresponding to different pH values, and D represents the ECL response results corresponding to different CuO@In2O3 concentrations.

[0104] Figure 13 Figure 1 shows the results of the ECL mechanism study. In this figure, A represents the ECL response of different materials in the K2S2O8-containing solution, with curves a, b, c, and d representing Gd MOF, Cu:Gd MOF, CuO / Cu:Gd MOF, and CuO@In2O3 / Cu:Gd MOF, respectively. In this figure, B represents the CV curves of different materials in the K2S2O8-containing solution, with curves a, b, c, and d representing Gd MOF, Cu:Gd MOF, CuO / Cu:Gd MOF, and CuO@In2O3 / Cu:Gd MOF, respectively. In this figure, C represents the ECL response of different materials in the K2S2O8-free solution, with curves a, b, and c representing Cu:Gd MOF, CuO / Cu:Gd MOF, and CuO@In2O3 / Cu:Gd MOF, respectively. In this figure, D represents the ECL spectra of Gd MOF (curve a) and Cu:Gd MOF (curve b). In this figure, E represents a schematic diagram of the ECL mechanism of the sensor.

[0105] Figure 14 The graph shows the detection performance results of the sensor, where A represents different concentrations of CA19-9 (ak: 0.5 mU / mL). -1 -100 U mL -1 The ECL response of the sensor is shown in Figure 5. B is the CA19-9 detection calibration curve, C is the CA19-9 detection calibration curve, D is the selectivity of the ECL immunosensor for different analytes, and the horizontal axis represents 1: blank, 2: NSE, 3: CEA, 4: NSE + CA19-9, 5: CEA + CA19-9, 6: CA19-9. E is the reproducibility of the ECL immunosensor, and the horizontal axis represents 1 to 6, which are 6 ECL immunosensors prepared by the preparation method in Example 5. F is the storage stability of the ECL immunosensor (n = 5). Detailed Implementation

[0106] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0107] The reagents used in the following examples include: gadolinium chloride hexahydrate (GdCl3·6H2O), 5-aminoisophthalic acid (5-AIPA), copper nitrate trihydrate (Cu(NO3)2·3H2O), copper chloride dihydrate (CuCl2·2H2O), and 3-aminopropyltriethoxysilane (APTES), all purchased from Shanghai Maclean Industrial Co., Ltd. Polyvinylpyrrolidone (PVP, Mw=10000), anhydrous ethanol, N,N-dimethylformamide (DMF), trimesic acid (H3BTC), indium nitrate hydrate (In(NO3)3·xH2O), and terephthalic acid (TPA) were provided by Sinopharm Chemical Reagents. All reagents used in this experiment were of analytical grade, and ultrapure water (18.25 MΩ·cm) was used throughout the experiments. -1 ).

[0108] The experimental instruments and equipment used in the following examples are shown in Table 1.

[0109] Table 1 lists the experimental instruments and equipment.

[0110]

[0111] Example 1

[0112] like Figure 1 As shown, a method for preparing a Gd MOF composite material includes the following steps:

[0113] S1. Dissolve 100 mg of polyether F127 in a mixed solution of 42 mL DMF and 6 mL H2O, then add 0.18 mmol of gadolinium chloride hexahydrate (GdCl3·6H2O) and 5-aminoisophthalic acid (5-AIPA) in the same amount as gadolinium chloride hexahydrate (GdCl3·6H2O), and sonicate until completely dissolved to obtain a mixed solution;

[0114] S2. The mixed solution obtained in S1 was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 120 °C for 12 h. After centrifugation, the precipitate was obtained. The precipitate was washed three times each with DMF and ethanol by centrifugation. Then, the precipitate was dried overnight under vacuum at 60 °C to obtain Gd MOF.

[0115] S3. Disperse 20 mg of Gd MOF obtained in S2 in 10 mL of water containing 0.2 mmol CuCl2·2H2O, and react at 80 ℃ for 4 h. Centrifuge and separate to obtain a precipitate. Wash the precipitate three times with water and dry it overnight under vacuum at 60 ℃ to obtain the Gd MOF composite material, namely Cu:Gd MOF.

[0116] Example 2

[0117] like Figure 1 As shown, a method for preparing an antibody-bound luminescent organism includes the following steps:

[0118] S1. Add 1.5 mg of Cu:Gd MOF prepared in Example 1 to 1 mL of water, disperse it evenly by ultrasonication, and then add 4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1 mg of N-hydroxysuccinimide (NHS) to obtain a mixture.

[0119] S2. The mixture obtained in S1 is shaken at 4 ℃ for 2 h for the first incubation, centrifuged, separated, and the precipitate is obtained. The precipitate is washed with water to remove unbound coupling agent and the intermediate product is obtained.

[0120] S3. Disperse the intermediate product obtained in S2 into 1 mL of PBS solution containing Ab2 (antibody for detecting carbohydrate antigen 19-9) (the concentration of Ab2 in the PBS solution is 1 µg / mL). -1 The sample was then shaken at 4 °C for 8 h, centrifuged, and separated to obtain a precipitate, which is the luminescent body that binds to the antibody, namely Cu:Gd MOF-Ab2. Cu:Gd MOF-Ab2 was dissolved in 1 mL of PBS (pH=7.4) solution to obtain Cu:Gd MOF-Ab2 PBS (pH=7.4) solution for later use.

[0121] Example 3

[0122] like Figure 1 As shown, a method for preparing a CuO composite material includes the following steps:

[0123] S1. Dissolve 0.3 g of polyvinylpyrrolidone (PVP) and 0.875 g of Cu(NO3)2·3H2O in 20 mL of DMF to obtain solution A;

[0124] S2. Dissolve 0.42 g of pyromellitic acid (H3BTC) in 20 mL of DMF to obtain solution B;

[0125] S3. Mix solution A obtained in S1 and solution B obtained in S2 and stir for 10 min, then transfer to a reaction vessel. React at 80 °C for 24 h, centrifuge, separate, and obtain precipitate. Wash the precipitate three times each with DMF and ethanol by centrifugation. Then dry the precipitate overnight under vacuum at 60 °C to obtain a blue powder, namely CuMOF.

[0126] S4. Dissolve 0.1 g of Cu MOF obtained in S3 in 30 mL of DMF, sonicate, add 0.4 mmol of In(NO3)3·4.5H2O and 0.4 mmol of terephthalic acid (TPA), mix well, and then transfer to a reaction vessel. React at 120 °C for 24 h, centrifuge, separate, and obtain precipitate. Wash the precipitate three times each with DMF, water and ethanol by centrifugation. Then dry the precipitate overnight under vacuum at 60 °C to obtain Cu MOF@In MOF.

[0127] S5. Place the Cu MOF@In MOF obtained in S4 into a muffle furnace, and then heat it at a rate of 5℃ / min. -1 The temperature was increased to 500℃ and then calcined at a constant temperature of 500℃ in a muffle furnace for 4 hours to obtain CuO@In2O3, i.e., CuO composite material.

[0128] Example 4

[0129] like Figure 1 As shown, a method for preparing CuO@In2O3-Ab1 includes the following steps:

[0130] S1. Dissolve 50 mg of CuO@In2O3 obtained in Example 3 in 10 mL of anhydrous ethanol, then add 0.1 mL of 3-aminopropyltriethoxysilane (APTES), and then react in an oil bath at 70 °C for 2 h. Centrifuge, separate, and obtain a precipitate. Dry and grind the precipitate to obtain CuO@In2O3-NH2.

[0131] S2. Disperse 1 mg of CuO@In2O3-NH2 obtained in S1 into 1 mL of PBS solution containing Ab1 (capture antibody for carbohydrate antigen 19-9), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS). Then, shake at 4°C for 8 h for a second incubation. Centrifuge and separate to obtain a precipitate. Wash the precipitate with PBS solution to remove unbound substances to obtain CuO@In2O3 bound to the antibody, which is the catalytic material for antibody binding, i.e., CuO@In2O3-Ab1. Disperse CuO@In2O3-Ab1 in 1 mL of PBS solution to obtain CuO@In2O3-Ab1 PBS (pH=7.4) solution for later use.

[0132] Example 5

[0133] like Figure 1 As shown, a method for preparing an immune sensor includes the following steps:

[0134] After polishing the glassy carbon electrode (GCE) with alumina powder, 10 µL of a 0.8 mg / mL solution was dropped onto the electrode surface. -1 The CuO@In2O3-Ab1 solution prepared in Example 4 was followed by the sequential addition of 3 µL of a 1% bovine serum albumin (BSA) solution in PBS (pH=7.4), 5 µL of CA19-9 antigen, and 6 µL of a 1.5 mg / mL solution. -1 The Cu:Gd MOF-Ab2 solution prepared in Example 2 was incubated in PBS (pH=7.4) at 4 °C for 2 h. After each layer modification, the electrode surface was rinsed with PBS (pH=7.4) to remove unbound substances or biomolecules, thus obtaining the ECL immunosensor.

[0135] Comparative Example 1

[0136] A method for preparing CuO includes the following steps:

[0137] S1. Dissolve 0.3 g of polyvinylpyrrolidone (PVP) and 0.875 g of Cu(NO3)2·3H2O in 20 mL of DMF to obtain solution A;

[0138] S2. Dissolve 0.42 g of pyromellitic acid (H3BTC) in 20 mL of DMF to obtain solution B;

[0139] S3. Mix solution A obtained in S1 and solution B obtained in S2 and stir for 10 min, then transfer to a reaction vessel. React at 80 °C for 24 h, centrifuge, separate, and obtain precipitate. Wash the precipitate three times each with DMF and ethanol by centrifugation. Then dry the precipitate overnight under vacuum at 60 °C to obtain a blue powder, namely CuMOF.

[0140] S4. Place the Cu MOF obtained in S3 into a muffle furnace, and then heat it at a rate of 5 °C / min. -1 The temperature was increased to 500 °C and then calcined at a constant temperature of 500 °C in a muffle furnace for 4 h to obtain CuO.

[0141] Comparative Example 2

[0142] A method for preparing In2O3 includes the following steps:

[0143] S1. Mix In(NO3)3·4.5H2O and 0.4 mmol of terephthalic acid (TPA) evenly, then transfer to a reaction vessel and react at 120 °C for 24 h. Centrifuge and separate to obtain a precipitate. Wash the precipitate three times each with DMF, water and ethanol by centrifugation. Then dry the precipitate overnight under vacuum at 60 °C to obtain In MOF.

[0144] S2. Place the In MOF obtained in S1 into a muffle furnace, and then heat it at a rate of 5 °C / min. -1 The temperature was increased to 500 °C and then calcined at a constant temperature of 500 °C in a muffle furnace for 4 h to obtain In2O3.

[0145] Detection and Analysis

[0146] 1) Characterization of Cu:Gd MOF

[0147] Elemental analysis was performed on the Gd MOF prepared in S2 and the Cu:Gd MOF prepared in S3 of Example 1 using X-ray photoelectron spectroscopy. Scanning electron microscopy (SEM) was used to analyze the Gd MOF prepared in S2 of Example 1. Transmission electron microscopy (TEM) was used to analyze the Gd MOF prepared in S2 of Example 1. The results are as follows: Figure 2 and Figure 3 As shown.

[0148] from Figure 2 As shown in A and B, Gd MOFs mainly consist of three elements: Gd, O, and C. Specifically, the satellite peaks appearing at 143.15 eV and 148.78 eV correspond to Gd 4d⁻¹, respectively. 5 / 2 and Gd 4d 3 / 2 And the Gd in the material is mainly composed of Gd 3+ The existence of Gd MOF in its current form demonstrates the successful synthesis of Gd MOF.

[0149] from Figure 2 As can be seen from C, Gd MOFs exhibit a tightly packed and smooth spherical morphology. Figure 2 The D image further reveals details of the microsphere, which is approximately 200 nm in size. From Figure 2 TEM characterization analysis of the E-molecules revealed that the Gd MOF is a hollow structure with a morphology similar to small bubbles.

[0150] from Figure 3 As shown in A and B, Cu doping of Gd MOF results in the appearance of copper in the XPS overall spectrum, and the binding energy positions of the Gd 4d orbitals show a significant shift compared to the undoped state. Further investigation of the fine spectrum of copper reveals… Figure 3As can be seen from C, the satellite peaks appearing at 935.16 eV and 954.74 eV correspond to Cu 2p, respectively. 3 / 2 and Cu2p 1 / 2 Furthermore, strong oscillating satellite peaks appeared at 945 and 965 eV, indicating that its valence state may be divalent. Further Auger spectrum testing was conducted, such as... Figure 3 As shown in Figure D, the least squares fitting method reveals a peak at 917.92 eV, indicating that copper is indeed present in the form of Cu. 2+ .

[0151] Fourier transform infrared spectroscopy (FTIR) was used to analyze the ligand 5-aminoisophthalic acid (5-AIPA), the Gd MOF prepared in Example 1 before and after doping, and the Cu:Gd MOF. Scanning electron microscopy (SEM) was used to analyze the Cu:Gd MOF prepared in S3 of Example 1. Energy dispersive X-ray spectroscopy (EDS) was used to perform elemental analysis of the Cu:Gd MOF prepared in S3 of Example 1. The results are as follows: Figure 4 As shown.

[0152] from Figure 4 A comparison of the infrared spectra of the ligand (5-AIPA) and the materials before and after doping (Gd MOF and Cu:Gd MOF) in Figure A shows that the characteristic absorption peaks did not change significantly, but the characteristic peaks after doping showed a slight red shift. Figure 4 As shown in B, copper doping increases the size of the microspheres, causes them to fracture noticeably, and results in a rougher surface. EDS elemental analysis was performed as follows: Figure 4 As shown in Figure C, the composite material contains four elements: Cu, Gd, C, and O, which are evenly distributed. This further proves the successful synthesis of MOF material and the successful doping of Cu.

[0153] 2) Photoluminescence properties of Cu:Gd MOF

[0154] The UV-Vis absorption spectra of the ligand 5-aminoisophthalic acid (5-AIPA), the Gd MOFs prepared in Example 1 before and after doping, and the Cu:Gd MOFs were measured using a UV-Vis spectrophotometer. The fluorescence excitation and emission spectra of the Gd MOFs prepared in Example 1 before and after doping, and the Cu:Gd MOFs were measured using a fluorescence spectrophotometer. The results are as follows: Figure 5 As shown.

[0155] from Figure 5Analysis of the UV-Vis absorption spectra of 5-AIPA shows a distinct absorption peak at 325 nm. The characteristic absorption peak of Gd MOF formed with 5-AIPA as a ligand is blue-shifted to 315 nm, which can be attributed to the π-π* absorption of the benzene ring in the ligand. The characteristic peak of Cu:Gd MOF further shows a slight blue shift (to 310 nm), presumably due to the influence of Cu doping.

[0156] from Figure 5 Analysis of spectra B and C reveals a strong emission peak in the broad band of 360–480 nm within the Gd MOF at an excitation wavelength of 315 nm. Based on similar spectra of the same ligands described in previous studies, this is speculated to be attributed to the fluorescence emission of the ligands, i.e., the π←π* transition occurring on the aromatic ring of the ligands. Furthermore, a small peak appears near 590 nm, which, according to previous reports of other lanthanides, may correspond to an intra-electron transition of the ion. Monitoring the excitation spectrum of Cu:Gd MOF shows a broad band, without the narrow maximum value observed due to the intra-electron excitation of Gd(III). This implies that the emission from the Gd(III) center preferentially occurs through ligand absorption; the excited ligand transfers energy to Gd(III), sensitizing it to emit light—an antenna effect. Figure 5 As can be seen from C, Cu:Gd MOF has two emission peaks at an excitation wavelength of 310 nm, with positions shifted to 380 ~ 450 nm and 600 ~ 650 nm, respectively. The emission peak in the 600 ~ 650 nm range is relatively stronger, indicating the sensitization effect of Cu.

[0157] 3) Characterization of CuO@In2O3

[0158] X-ray diffraction (XRD) was used to analyze the CuO@In₂O₃ prepared in Example 3, the CuO prepared in Control Example 1, and the In₂O₃ prepared in Control Example 2. Elemental analysis was performed on the CuO@In₂O₃ prepared in Example 3 using X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was used to analyze the Cu MOF prepared in S3, CuMOF@In MOF prepared in S4, and CuO@In₂O₃ prepared in S5 of Example 3. Transmission electron microscopy (TEM) was used to analyze the CuO@In₂O₃ prepared in S5 of Example 3. EDS elemental analysis was performed on the CuO@In₂O₃ prepared in Example 3 using a combination of scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The results are as follows: Figure 6 and Figure 7 As shown.

[0159] from Figure 6As can be seen from A and B, the diffraction peaks of the synthesized CuO at 32.5°, 35.5°, 38.7°, and 48.6° correspond to the (110), (002), (111), and (-202) crystal planes in the standard card (PDF#80-1917), respectively. The In2O3 exhibits distinct diffraction peaks at 21.5°, 30.6°, 35.5°, 51.0°, and 60.7°, which are attributed to the (211), (222), (400), (440), and (622) crystal planes (PDF#71-2194), respectively. Furthermore, the XRD pattern of the composite material CuO@In2O3 simultaneously shows the strongest characteristic peaks of both pure substances, thus proving the successful synthesis of the material.

[0160] At the same time, from Figure 6 The elemental composition of the CuO@In2O3 composite material in C also confirms the successful synthesis of the material. Specifically, from Figure 6 As can be seen from D, the Cu 2p orbit can be divided into two satellite peaks, corresponding to Cu 2p 3 / 2 (933.01 eV) and Cu 2p 1 / 2 (952.73 eV). The graph shows three strong oscillating peaks, further confirming that Cu's valence state is indeed +2. Meanwhile, from... Figure 6 As can be seen from E, the satellite peaks of In at 444.39 eV and 451.94 eV correspond to its 3d E values, respectively. 5 / 2 and 3D 3 / 2 The orbit, its existence form is In 3+ .from Figure 6 From F, we can see that the fine spectrum of the O 1s orbital has peaks around 529 eV and 531 eV, indicating that the O element is represented by O. 2- It exists in the form of, but with different metal ions (Cu). 2+ and In 3+ Coordination is performed.

[0161] The morphology of the material was further characterized using SEM. Figure 7 As can be seen from A, the synthesized Cu MOF possesses a complete octahedral structure with a smooth surface and sharp edges. From Figure 7 As can be seen from B, in the further synthesized Cu MOF@InMOF, In MOF grows along its outer surface, the octahedrons are clearly covered, and the surface roughness is significantly increased. From Figure 7 As can be seen from C, further calcination yields an octahedron with a certain degree of collapse, consisting of a layered structure, which is CuO@In2O3. Figure 7 As can be seen from D, the internal structure and details of CuO@In2O3 material can be analyzed and calculated using high-resolution transmission electron microscopy (TEM) images, such as... Figure 7 As shown in E, distinct lattice spacings of CuO and In2O3 are obtained, which are 0.25 nm and 0.29 nm, respectively, corresponding to the (002) crystal plane of CuO and the (222) crystal plane of In2O3. Furthermore, from... Figure 7 The presence and distribution of Cu, In, and O observed in the EDS mapping spectrum of F fully demonstrate the successful synthesis of CuO@In2O3 material.

[0162] 4) Investigation on the catalytic performance of CuO@In2O3

[0163] The glassy carbon electrode (GCE) was polished with alumina powder, and then 10 µL of a 0.8 mg / mL solution was dropped onto the electrode surface. -1 The CuO aqueous solution prepared in Example 1 was used to modify the electrode surface with a single layer, and then incubated at 4 °C for 2 h. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, and the CuO electrode was obtained.

[0164] The glassy carbon electrode (GCE) was polished with alumina powder, and then 10 µL of a 0.8 mg / mL solution was dropped onto the electrode surface. -1 The CuO@In2O3 aqueous solution prepared in Example 3 was used to modify the electrode surface as a monolayer. Then, it was incubated at a temperature of 4 °C for 2 h. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, thus obtaining the CuO@In2O3 electrode.

[0165] An ECL immunosensor was constructed using Ag / AgCl and Pt as the reference and counter electrodes, respectively, and the aforementioned CuO and CuO@In2O3 electrodes as the working electrodes. The sensors were then tested at 5.0 mmol / L... -1 Cyclic voltammetry (CV) was performed in a K3[Fe(CN)6] solution with the following parameters set: scan rate of 25 ~ 225 mV s. -1 (n = 3). The results of cyclic voltammetry (CV) testing and the linear relationship between peak current and sweep rate are as follows: Figure 8 and Figure 9 As shown.

[0166] According to the Randles-Sovcik equation shown in equation (Ⅰ) and Figure 8 and Figure 9 The electrochemical active areas of CuO and CuO@In2O3 were calculated using cyclic voltammetry (CV) results.

[0167]

[0168] In the Randles-Sovcik equation shown in equation (Ⅰ), I represents the reduction peak current, and A represents the electrochemical active area to be calculated (cm²). 2 D represents the diffusion coefficient of K3[Fe(CN)6] at room temperature, which is approximately 6.70 × 10⁻⁶. -6 cm 2 s -1 n1 represents the number of electrons transferred, n2 represents the scan rate, and c represents the concentration of the K3[Fe(CN)6] solution.

[0169] like Figure 8 and Figure 9 As can be seen from the results, the linear fitting equations based on the CV test results are I = 228.98n. 1 / 2 +9.61 and I = 273.17n 1 / 2 +2.66. Based on the slope of the linear fitting equation, the A values ​​of CuO and CuO@In2O3 can be calculated to be 6.58 mm. 2 and 7.85 mm 2 This indicates a certain increase in the electrochemical active area. Furthermore, the stronger current response value also indicates an increase in electron transfer rate and enhanced catalytic performance.

[0170] 5) Electrochemical characterization of biosensors with different numbers of modified layers

[0171] 5.1) Polish the glassy carbon electrode (GCE) with alumina powder, then drop 10 µL of a 0.8 mg / mL solution onto the electrode surface. -1 The CuO@In2O3-Ab1 PBS (pH 7.4) solution prepared in Example 4 was used to perform monolayer modification on the electrode surface, and then incubated at 4 °C for 2 h. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, resulting in the CuO@In2O3-Ab1 / GCE electrode (b).

[0172] 5.2) Polish the glassy carbon electrode (GCE) with alumina powder, then drop 10 µL of a 0.8 mg / mL solution onto the electrode surface. -1The CuO@In2O3-Ab1 PBS (pH 7.4) solution prepared in Example 4 was then used to add 3 µL of 1% bovine serum albumin (BSA) PBS (pH 7.4) solution to the electrode surface to perform two-layer modification of the electrode surface. The electrode was then incubated at 4 °C for 2 h. After each layer modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, resulting in the BSA / CuO@In2O3-Ab1 / GCE electrode (c).

[0173] 5.3) Polish the glassy carbon electrode (GCE) with alumina powder, then drop 10 µL of a 0.8 mg / mL solution onto the electrode surface. -1 The CuO@In2O3-Ab1 PBS (pH 7.4) solution prepared in Example 4 was then used to sequentially add 3 µL of 1% bovine serum albumin (BSA) PBS (pH 7.4) solution and 5 µL of CA19-9 antigen to the electrode surface to perform a three-layer modification. The electrode surface was then incubated at 4 °C for 2 h. After each layer modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, resulting in the CA19-9 / BSA / CuO@In2O3-Ab1 / GCE electrode (d).

[0174] Ag / AgCl and Pt were used as the reference electrode and counter electrode, respectively. An unmodified glassy carbon electrode (GCE) (a), CuO@In2O3-Ab1 / GCE electrodes prepared with different modification layers (b), BSA / CuO@In2O3-Ab1 / GCE electrodes (c), CA19-9 / BSA / CuO@In2O3-Ab1 / GCE electrodes (d), or the ECL immunosensor constructed in Example 5 (e) were used as working electrodes for electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) tests. The parameters set during the tests were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of -0.20 ~ 0.6 V, and scan rate of 0.1 V s. -1 The test solution was 10 mL of KCl (0.1 mol / L). -1 ) and K2S2O8 (120 mmol L -1 A mixed PBS solution (pH 7.4). Results are as follows. Figure 10 and Figure 11 As shown.

[0175] Figure 10 and Figure 11In the diagram, the black curve corresponds to the measurement results of the unmodified glassy carbon electrode (a) as the working electrode; the red curve corresponds to the measurement results of the monolayer modified CuO@In2O3-Ab1 / GCE electrode (b) as the working electrode; the blue curve corresponds to the measurement results of the bilayer modified BSA / CuO@In2O3-Ab1 / GCE electrode (c) as the working electrode; the green curve corresponds to the measurement results of the trilayer modified CA19-9 / BSA / CuO@In2O3-Ab1 / GCE electrode (d) as the working electrode; and the purple curve corresponds to the measurement results of the four-layer modified ECL immunosensor (e) constructed in Example 5 as the working electrode, i.e., the measurement results of the ECL immunosensor constructed in Example 5 as the working electrode.

[0176] from Figure 10 It can be seen that after modifying the electrode surface with CuO@In2O3-Ab1, the peak current of its cyclic voltammetry (CV) curve is significantly lower than that of the bare glassy carbon electrode (GCE), and the redox potential also changes. Furthermore, with the layer-by-layer modification of BSA, CA19-9, and Cu:Gd MOF-Ab2, electron transfer is hindered, and the peak current gradually decreases, proving the successful construction of the sensor. Meanwhile, from... Figure 11 The results of the electrochemical impedance spectroscopy (EIS) test show that, due to the poor conductivity of biomacromolecules and their conjugates, the impedance value gradually increases during the layer-by-layer modification process, which also verifies the successful construction of the sensor.

[0177] 6) Performance testing of the ECL immune sensor

[0178] Effect of K2S2O8 concentration

[0179] ECL testing employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. First, the Cu:Gd MOF prepared in Example 1 was added to deionized water to prepare a mixture with Cu:Gd MOF concentrations of 1.5 mg / mL. This mixture was then dropwise added to a glassy carbon electrode (GCE) to form a modified layer, which was used as the working electrode. The parameters set during testing were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of 0 ~ 0.7 V, and scan rate of 0.1 V s. -1 The ECL response was tested using 10 mL of PBS (pH=7.4) solutions with different concentrations of K2S2O8 as the substrate. The results are as follows: Figure 12 As shown in A.

[0180] Effect of Cu:Gd MOF concentration

[0181] ECL testing employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. First, the Cu:Gd MOF prepared in Example 1 was added to deionized water to prepare mixed solutions with Cu:Gd MOF concentrations of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. These mixed solutions were then dropwise added to a glassy carbon electrode (GCE) to form a modified layer, which was used as the working electrode. The test substrate consisted of 10 mL of 120 mmol / L glassy carbon dioxide. -1 The ECL response of K2S2O8 in PBS (pH=7.4) was as follows: Figure 12 As shown in B.

[0182] The effect of pH

[0183] ECL testing employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. First, the Cu:Gd MOF prepared in Example 1 was added to deionized water to prepare a mixture with Cu:Gd MOF concentrations of 1.5 mg / mL. This mixture was then dropwise added to a glassy carbon electrode (GCE) to form a modified layer, which was used as the working electrode. The testing parameters were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of 0 ~ 0.7 V, and scan rate of 0.1 V s. -1 The test solutions were 10 mL of 120 mmol / L solution at different pH values. -1 The ECL response of K2S2O8 in PBS solution was as follows: Figure 12 As shown in C.

[0184] Effect of CuO@In2O3 concentration

[0185] ECL testing employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. 10 µL of each electrode with a concentration of 0.1 mg / mL was added to the electrode surface. -1 0.3 mg mL -1 0.5 mg mL -1 0.8 mg mL -1 1.0 mg mL -1 The CuO@In2O3 aqueous solution prepared in Example 3 was then supplemented with 6 µL of a 1.5 mg / mL solution. -1 The Cu:Gd MOF aqueous solution prepared in Example 2 forms two modified layers, which are used as the working electrode. The test substrate is 10 mL of 120 mmol / L. -1 The ECL response of K2S2O8 in PBS (pH=7.4) was as follows: Figure 12As shown in D.

[0186] from Figure 12 As can be seen from the data, the optimal conditions selected were a K₂S₂O₈ concentration of 120 mmol / L in the K₂S₂O₈ aqueous solution. -1 The concentration of Cu:Gd MOF in the Cu:Gd MOF aqueous solution was 1.5 mg / mL. -1 The pH value is 7.4, and the concentration of CuO@In2O3 in the CuO@In2O3 aqueous solution is 0.8 mg / mL. -1 .

[0187] 7) Research on ECL mechanisms

[0188] The aforementioned studies on the photoluminescence properties of Gd MOF and Cu:Gd MOF demonstrated the existence of energy transfer from the ligand to the central ion (antenna effect) and the sensitization effect of Cu doping in MOF, which effectively enhances the luminescence intensity. Although the excitation methods of photoluminescence and electroluminescence are different, the energy transition mode and the result of photons emitted from the excited state returning to the ground state generally do not change much. Therefore, further research is needed on the ECL mechanism of the materials.

[0189] Specifically:

[0190] The glassy carbon electrode (GCE) was polished with alumina powder, and then 10 µL of a 1.5 mg / mL solution was dropped onto the electrode surface. -1 The Gd MOF aqueous solution prepared in S2 of Example 1 was used to modify the electrode surface as a monolayer. Then, it was incubated at 4 °C for 2 h. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, and the Gd MOF electrode was obtained.

[0191] The glassy carbon electrode (GCE) was polished with alumina powder, and then 10 µL of a 1.5 mg / mL solution was dropped onto the electrode surface. -1 The Cu:Gd MOF aqueous solution prepared in Example 1 was used to modify the electrode surface as a monolayer. The electrode was then incubated at 4 °C for 2 h. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, thus obtaining the Cu:Gd MOF electrode.

[0192] The glassy carbon electrode (GCE) was polished with alumina powder, and then 10 µL of a 0.8 mg / mL solution was sequentially added to the electrode surface. -1 The CuO aqueous solution prepared in Comparative Example 1 and 10 µL of a solution with a concentration of 1.5 mg / mL were compared. -1The Cu:Gd MOF aqueous solution prepared in Example 1 was used to modify the electrode surface in two layers. Then, it was incubated at 4 °C for 2 h. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, thus obtaining the CuO / Cu:Gd MOF electrode.

[0193] The glassy carbon electrode (GCE) was polished with alumina powder, and then 10 µL of a 0.8 mg / mL solution was sequentially added to the electrode surface. -1 The CuO@In2O3 aqueous solution prepared in Example 3 and 10 µL of a solution with a concentration of 1.5 mg / mL -1 The Cu:Gd MOF aqueous solution prepared in Example 1 was used to modify the electrode surface in two layers. Then, it was incubated at 4 °C for 2 h. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, thus obtaining the CuO@In2O3 / Cu:Gd MOF electrode.

[0194] Ag / AgCl and Pt were used as the reference and counter electrodes, respectively, and the Gd MOF electrode, Cu:Gd MOF electrode, CuO / Cu:Gd MOF electrode, and CuO@In2O3 / Cu:Gd MOF electrode prepared above were used as the working electrodes. The parameters set during the test were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of -2 ~ 0 V, and scan rate of 0.1 V s. -1 .

[0195] At a pH of 7.4, 10 mL of solution had a concentration of 120 mmol / L. -1 ECL testing was performed on K2S2O in PBS solution, and the results are as follows: Figure 13 As shown in A.

[0196] from Figure 13 As shown in Figure A, curve a, representing the Gd MOF, exhibits a relatively weak signal intensity (1500 au), while curve b, representing the Cu:Gd MOF, shows a signal enhancement of 3 times (6000 au). After modification with CuO, the signal of curve c, representing the CuO / Cu:Gd MOF, is further enhanced, while after modification with CuO@In2O3, the signal of curve d, representing the CuO@In2O3 / Cu:Gd MOF, is the strongest, reaching almost 18000 au.

[0197] The test results in pure PBS solution are as follows: Figure 13 As shown in C.

[0198] from Figure 13As shown in Figure C, the ECL signals of Cu:Gd MOF represented by curve a, CuO / Cu:Gd MOF represented by curve b, and CuO@In2O3 / Cu:Gd MOF represented by curve c are all very low and do not show significant enhancement. This indicates that K2S2O8 is an indispensable co-reactant for ECL, and the catalytic effect of CuO and CuO@In2O3 only acts on K2S2O8, not Cu:Gd MOF.

[0199] Further CV testing was conducted, and the results were as follows: Figure 13 As shown in B.

[0200] from Figure 13 As can be seen from curve B, curve a, representing Gd MOF, has a relatively broad K2S2O8 reduction peak, while curve b, representing Cu:Gd MOF, shows a change compared to this curve, with a significant positive shift in the K2S2O8 reduction peak. This indicates that copper doping has an effect, promoting the reduction of K2S2O8. Furthermore, from... Figure 13 As can be seen from B, the reduction peaks of the CV curves of CuO / Cu:Gd MOF represented by curve c and CuO@In2O3 / Cu:Gd MOF represented by curve d also shifted slightly positively, indicating that the change in the valence state of Cu and In in the material catalyzed the reduction of K2S2O8 to a certain extent and enhanced the ECL signal.

[0201] Comparison of ECL spectra of Gd MOF represented by curve a and Cu:Gd MOF represented by curve b, for example Figure 13 As shown in D.

[0202] from Figure 13 As can be seen from D, the emission wavelength range of both did not change significantly, but the emission intensity increased significantly, indicating that Cu doping did not change the ECL properties of Gd MOF, and it is highly likely that it acted on the co-reactant K2S2O8.

[0203] Based on the above results, it can be concluded that the ECL mechanism of the sensor is related to the light enhancement mechanism as follows: Figure 13 As shown in E. Specifically, as the potential decreases, S2O8 2- Gaining an electron produces a small amount of and SO4 2- (Path 1). Afterwards, Cu 2+ / Cu + and In 3+ / In 2+ Two redox pairs accelerated The formation of Cu. In short, Cu 2+ / In 3+ Electrons can be trapped on the electrode surface to generate Cu. + / In 2+Then with S2O8 2- The reaction yielded a large amount ( Figure 13 The orange color in E (path 2). Furthermore, OH can be generated through a series of redox reactions. • Afterwards, the generated OH... • With S2O8 2- Further reactions generate more ( Figure 13 (Blue in E, path 3). For the ECL emission process, Cu:Gd MOF first gains electrons at the electrode surface to generate Cu. + and Then Cu + Promote S2O8 2- The restoration generates more ( Figure 13 The yellow part in E (path 4). and The reaction produces Gd MOF*, and finally, the generated Gd MOF* transitions back to the ground state and produces a photon. Figure 13 The green color in E (ECL emission). The specific reaction mechanism is as follows:

[0204] Path 1

[0205]

[0206] Path 2

[0207]

[0208] Path 3

[0209]

[0210] Path 4

[0211]

[0212] ECL launch

[0213]

[0214] 8) Sensor detection performance

[0215] Under optimal testing conditions, namely the ECL immunosensor prepared in Example 5, the electrolyte concentration was 120 mmol / L. -1 The test was conducted using K2S2O8 in PBS (pH 7.4) solution to obtain ECL intensity-time curves for different concentrations of CA19-9. The results are as follows: Figure 14As shown in Figure A, the ECL signal increases with the increase of CA19-9 concentration.

[0216] Further, the calibration curve for CA19-9 detection was obtained, from... Figure 14 As can be seen from B, at 0.5 mU / mL -1 ~ 100U mL -1 Within the range, ECL intensity and the logarithm of CA19-9 concentration showed a good linear relationship (I = 2162 lg c + 4456, R). 2 = 0.997). Compared with existing sensors (see Table 2), the constructed ECL immunosensor achieves sensitive analysis of CA19-9 over a wider linear range, and the calculated limit of detection is as low as 82.3 µU / mL. -1 (S / N = 3).

[0217] To verify the stability of the sensor, 12 consecutive cycles of scanning were performed on one electrode, and the results were as follows: Figure 14 As shown in C, the peak intensity of ECL in each period has small differences, and its RSD is calculated to be 2.06%, indicating that the sensor has good stability.

[0218] The selectivity of the ECL immune sensor was studied by testing the ECL response to CA19-9 and other interfering agents. The results are as follows: Figure 14 As shown in D. From Figure 14 As can be seen from D, the ECL responses of neuron-specific enolase (NSE) and carcinoembryonic antigen (CEA) are close to the ECL signals of the blank sample, while the ECL response of the mixed sample is almost identical to that of CA19-9 (5 U / mL). -1 The presence of CA19-9+ (10 times the concentration of interfering substances) indicates that the sensor has good selectivity.

[0219] Under the same conditions, the ECL signals of six ECL immunosensors prepared using the method described in Example 5, respectively, were detected as working electrodes to study the reproducibility of the biosensors. The results are as follows: Figure 14 As shown in E, the results obtained have only minor differences.

[0220] The storage stability of the ECL immune sensor was tested, and the results are as follows: Figure 14 As shown in F, after 12 days of storage at 4°C, the ECL signal only decreased by 22.6% of the initial value, demonstrating good storage stability.

[0221] In summary, this invention fully demonstrates that the biosensor constructed in this invention exhibits excellent ECL performance and can be used for trace detection of the target CA19-9.

[0222] Table 2 compares the performance of CA19-9 detection with other literature.

[0223]

[0224] 9) Serum sample analysis

[0225] Human serum samples were selected as the initial samples. Different concentrations of CA19-9 were added to the initial human serum samples using a standard addition-recovery method. After five tests on each spiked sample using the ECL sensor prepared in Example 5, the average detection concentration of the target was calculated. The recovery rate and RSD were then calculated based on the obtained detection concentrations to analyze the accuracy and precision of the biosensor in serum sample detection. As shown in Table 3, the recovery rate and RSD ranged from 98.6% to 102% and 0.86% to 1.07%, respectively, indicating that the ECL biosensor constructed in this invention has good accuracy and precision in serum sample analysis and has certain clinical application value.

[0226] Table 3 shows the application of the constructed ECL immunosensor in serum sample analysis.

[0227]

[0228] In summary, this invention investigated the electrochemiluminescence behavior of Gd MOF and Cu:Gd MOF. As a lanthanide MOF material, Gd MOF exhibits an antenna effect, enabling energy transfer from ligands to the metal center ion, and thus possesses excellent PL and ECL properties. Cu doping effectively shortens the charge transfer distance and accelerates the electron transfer rate, significantly enhancing the electrochemiluminescence intensity of Gd MOF. Furthermore, CuO@In2O3 was synthesized as a co-reaction promoter to construct Cu:Gd MOF / S2O8. 2- The / CuO@In2O3 ternary system further enhanced the ECL signal. By monitoring changes in ECL intensity, the target analyte CA19-9 can be quantitatively detected, i.e., CA19-9 can be quantified through ECL signal enhancement. This study demonstrates the potential of Gd MOFs for electrochemiluminescence, which to some extent broadens the application and development of metal-doped lanthanide MOFs in the ECL field. Furthermore, the constructed enhanced ECL immunosensor achieves the goal of high-level analysis using biosensors.

[0229] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A method for preparing a luminescent body bound to an antibody, characterized in that Includes the following steps: Gd MOF composite material was added to water, followed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide for the first incubation. Then it was dispersed in PBS solution containing antibody II and incubated for a further period to obtain a luminescent body that binds to the antibody. The preparation method of the Gd MOF composite material includes the following steps: Polyether F127 was dissolved in a mixed solvent, and then gadolinium salt and 5-aminoisophthalic acid were added. A first heating reaction was carried out to obtain Gd MOF. Gd MOF was dispersed in a copper salt solution and subjected to a second heating reaction to obtain a Gd MOF composite material, namely Cu:Gd MOF.

2. The method of claim 1, wherein the luminescent binding antibody is prepared by the steps of: The temperature of the first heating reaction is 120~130 ℃, and the time is 12~13 h; And / or, the temperature of the second heating reaction is 80~90℃, and the time is 4~5h; And / or, the ratio of the polyether F127, gadolinium salt, and 5-aminoisophthalic acid is 100 mg: 0.18 mmol: 0.18 mmol; And / or, the ratio of the Gd MOF to the copper salt is 20 mg: 0.2 mmol; And / or, the gadolinium salt is selected from one or both of gadolinium chloride and gadolinium chloride hexahydrate; And / or, the copper salt is selected from one or both of copper chloride and copper chloride dihydrate; And / or, the mixed solvent is selected from a mixture of N,N-dimethylformamide and water; And / or, the copper salt solution is an aqueous solution of copper salt.

3. A luminescent body binding an antibody, characterized in that It is prepared by the preparation method described in claim 1 or claim 2.

4. The method for preparing an antibody-bound luminescent organism according to claim 1, characterized in that, The antibody II is selected from Ab2 antibody; And / or, the first incubation temperature is 4 °C and the time is 2 h; And / or, the continued incubation temperature is 4 °C and the time is 8 h.

5. An immune sensor, characterized in that, Includes a luminescent bioluminescent device that binds to an antibody, prepared by the method described in claim 1 or claim 2.

6. A method for preparing an immune sensor as described in claim 5, characterized in that, Includes the following steps: CuO@In2O3 was dissolved in a second organic solvent, and then 3-aminopropyltriethoxysilane was added. A fifth heating reaction was carried out to obtain CuO@In2O3-NH2. CuO@In2O3-NH2 was dispersed in a PBS solution containing antibody I, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then incubated for a second time to obtain antibody-bound CuO@In2O3. A CuO@In2O3 solution containing the bound antibody was dropped onto the electrode surface, followed by the sequential addition of bovine serum albumin solution, CA19-9 antigen, and the luminescent solution containing the bound antibody to the electrode surface for a third incubation to obtain the immunosensor.

7. The method for preparing the immune sensor according to claim 6, characterized in that, The antibody I is selected from Ab1 antibody; And / or, the second organic solvent is selected from anhydrous ethanol; And / or, the ratio of CuO@In2O3 to 3-aminopropyltriethoxysilane is 500 mg: 1 mL.

8. The method for preparing the immune sensor according to claim 6, characterized in that, The fifth heating reaction is carried out at a temperature of 70 °C for 2 h. And / or, the second incubation temperature is 4 °C and the time is 8 h; And / or, the third incubation temperature is 4 °C and the time is 2 h.

9. An immunosensor prepared by the method according to any one of claims 6 to 8, used as an ECL immunosensor, characterized in that, The immunosensor is used to detect carbohydrate antigen 19-9.

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